Protein kinase C-dependent inhibition of Kir channels

Kir 通道的蛋白激酶 C 依赖性抑制

• 批准号: 6642457
• 负责人: Diomedes E. Logothetis
• 金额: $ 4.14万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6642457
• 关键词: CHO cells; Xenopus oocyte; binding proteins; biological signal transduction; confocal scanning microscopy; electrophysiology; hydrolysis; kinase inhibitor; muscarine; phosphatidylinositols; polymerase chain reaction; potassium channel; protein isoforms; protein kinase C; protein protein interaction; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant) It has been recently appreciated that a common characteristic of members of the inwardly rectifying K+ (Kir) channel family is that they are all activated by phospatidylinositol-bis-phosphate (PIP2). Differences in channel-PIP2 interactions have been described among specific Kir members, both biochemically (Huang et al., 1998) and functionally (Huang et al., 1998; Zhang et al., 1999; Lopes et al., 2002). There seem to be Kir channels that show either relatively strong, intermediate or weak interactions with PIP2. Certain Kir channels are inhibited by PIP2 hydrolysis. The strength of channeI-PIP2 interactions correlates with the extent of channel activity and the degree of channel inhibition caused by signals that lead to PIP2 hydrolysis. Thus, channels that interact weakly with PIP2 are inhibited the most by PIP2 hydrolysis, while channels that interact strongly with PIP2 are not inhibited by PIP2 hydrolysis. Channel modulation by PKC affects channel activity in a manner dependent on channel-PIP2 interactions. Thus, in a wild-type channel showing PKC-mediated inhibition of activity, mutations strengthening channel-PIP2 interactions can attenuate or abolish the effect of PKC. Similarly, in a channel that interacts strongly with PIP2 and therefore lacks PKC-dependent inhibition, mutations that weaken channeI-PIP2 interactions can render the channel inhibitable by activated PKC. The PKC-mediated inhibition could be obtained in heterologous systems such as in Xenopus oocytes but not in others, such as the CHO mammalian cells. The current proposal aims to find answers to the following two major questions. (a) Are there specific PKC isoforms that are needed to reconstitute the PKC-mediated current inhibition in certain cell systems where the effect is absent? Are there specific PKC adaptor proteins involved in mediating the PKC effects? (b) To what extend is the muscarinic induced inhibition of Kir currents due to PKC mediated effects? Does PKC modulation of Kir channel activity weaken channel-PIP2 interactions? The current proposal expands and enhances the parent grant HL59949. In the parent grant our emphasis is to identify the PKC-dependent phosphorylation sites either on the channel protein itself or associated proteins and to study their relationship to channeI-PIPz interacting sites. In the current grant the focus is to identify the specific PKC isoforms and/or adaptor proteins involved in the PKC-mediated inhibition and to determine the relative contribution to the ACh-induced current inhibition by PKC versus the decrease in direct channel-PIP2 interactions that result due to the PIP2 hydrolysis.

描述（由申请人提供） 最近已经认识到，内向整流K+（Kir）通道家族的成员的共同特征是它们都被磷脂酰肌醇二磷酸（PIP 2）激活。通道-PIP 2相互作用的差异已经在特定的Kir成员之间进行了描述，无论是在生物化学上（Huang等人，1998）和功能上（Huang等人，1998; Zhang等人，1999; Lopes等人，2002年）。似乎存在显示与PIP 2的相对强、中等或弱相互作用的Kir通道。某些Kir通道被PIP 2水解抑制。通道I-PIP 2相互作用的强度与通道活性的程度和由导致PIP 2水解的信号引起的通道抑制的程度相关。因此，与PIP 2弱相互作用的通道被PIP 2水解抑制最多，而与PIP 2强相互作用的通道不被PIP 2水解抑制。通过PKC的通道调节以依赖于通道-PIP 2相互作用的方式影响通道活性。因此，在显示PKC介导的活性抑制的野生型通道中，增强通道-PIP 2相互作用的突变可以减弱或消除PKC的作用。类似地，在与PIP 2强烈相互作用且因此缺乏PKC依赖性抑制的通道中，减弱通道I-PIP 2相互作用的突变可使通道可通过活化的PKC而被激活。PKC介导的抑制可以在异源系统中获得，例如在非洲爪蟾卵母细胞中，但在其他系统中不能获得，例如CHO哺乳动物细胞。本提案旨在为以下两个主要问题找到答案。(a)在某些不存在这种效应的细胞系统中，是否需要特定的PKC亚型来重建PKC介导的电流抑制？是否有特定的PKC衔接蛋白参与介导PKC的作用？(b)毒蕈碱诱导的Kir电流抑制到何种程度是由于PKC介导的作用？PKC对Kir通道活性的调节是否减弱了通道-PIP 2的相互作用？目前的建议扩大和加强母补助金HL 59949。在本研究中，我们的重点是鉴定通道蛋白本身或相关蛋白上的PKC依赖性磷酸化位点，并研究它们与通道I-PIPz相互作用位点的关系。在目前的资助重点是确定具体的PKC亚型和/或衔接蛋白参与PKC介导的抑制，并确定相对的贡献，乙酰胆碱诱导的电流抑制PKC与减少直接通道PIP 2相互作用的结果，由于PIP 2水解。